The experimental goals of the proposed studies are to determine: 1) what features of a mammalian messenger RNA are important for its recognition by ribosomes; and 2) what the precise rate-limiting event is in the initiation of translation in a mammalian cell. Since it is well documented that each molecule of beta globin mRNA initiates protein synthesis at a rate 40-50 percent faster than alpha mRNA within the reticulocyte, the globin mRNAs will primarily be used in these studies as a comparative system for studying translational control in a terminally differentiated mammalian cell. My previous studies on nucleotide sequence analysis of 5'-noncoding regions of mouse and rabbit alpha and beta globin mRNAs did not reveal crucial similarities or differences which could possibly explain these aspects of the initiation process. More recently, however, secondary structure has been examined in the globin mRNAs. By using both single-strand specific S1 nuclease and base specific T1 nuclease as structure probes on 5'-32P-end labeled mouse and rabbit globin mRNA, followed by analysis on polyacrylamide sequencing gels, potentially important structural differences in the initiator codon regions between these alpha and beta mRNAs have been elucidated. Since this data suggests a possible structural specificity in controlling the differential rates of initiation of these two mRNA molecules, these studies will be extended by determining the complete secondary structures of mouse alpha and beta mRNA using chemical modification, psoralen cross-linking and nuclease susceptibility as structure probes. With strucutre data for the entire molecules, one could evaluate potential structure-function relationships during translational control with greater confidence. The strucutral analyses of initiator regions will be extended to the goat globin mRNAs as another comparative system, as well as STNV RNA, which is a plant viral RNA known to initiate fairly efficiently without a m7G5'ppp5'-cap structure. These studies will be complemented with ribosome binding studies in reticulocyte lysates to specifically correlate structure with their function.